With the advent of hybrid DNA technology and the explosion in the ability to isolate, purify, and assay a wide variety of natural products, both polypeptides and nucleic acids, there is an increasing need for rapid and efficient methods of preparing and purifying oligomers of amino acids and nucleic acids.
With nucleic acids, it is typically necessary to synthesize sequences for use as linkers, adapters, synthetic genes and synthetic regulatory sequences, as well as for use as probes, primers, and the like. Many procedures have been developed for producing oligomers of nucleotides. These procedures for the most part rely on initial attachment of a first nucleotide to a solid support by a selectively cleavable linkage, followed by sequential addition of subsequent nucleotide units, with each addition involving a number of chemical reactions.
The two primary methods of oligonucleotide synthesis which are well-established in the art are the so-called "phosphotriester" and "/phosphoramidite" methods (described at some length in the references cited below). In the most prevalent schemes for both methods, the oligonucleotide chain grows by nucleophilic attack of the 5'-OH of the immobilized oligomer on an activated 3,-phosphate or phosphoramidite function of a soluble 5,-protected nucleotide building block. Other key steps include the acid deprotection of the 5,-O-(4,4,dimethoxytrityl) group (DMTr) in the phosphotriester method, and, in the phosphoramidite process, the oxidation of the phosphite triester to the phosphate triester.
Other methods of oligonucleotide synthesis are also known, including 5' to 3' syntheses which use a cyanoethyl phosphate protecting group (De Napoli et al., Gazz. Chim. Ital. 114:65 (1984); Rosenthal et al., Tetrahedron Lett. 24:1691 (1983); Belagaje and Brush, Nucleic Acids Res. 10:6295 (1977)) and solution phase 5' to 3' syntheses (e.g., Hayatsu and Khorana, J. Amer. Chem. Soc. 89:3880 (1967); Gait and Sheppard, Nucleic Acids Res. 4:1135 (1977); Cramer and Koster, Angew. Chem. Int. Ed. Engl. 7:473 (1968); and Blackburn et al., J. Chem. Soc. C, 2438 (1967))
After completion of oligonucleotide synthesis and deprotection of the product, the free 5'-OH group of the oligonucleotide must be phosphorylated or phosphitylated for use in most biological processes. Also, phosphorylation or phosphitylation on the 3'-OH function is typically necessary to generate oligonucleotides in a form that can be purified, stored and/or commercialized. See Sonveaux, Bioorganic Chem. 14:274,294 (1986) The present invention is directed to compounds which are useful in phosphorylating and phosphitylating both 3' and 5' hydroxyl moieties.
5' -phosphorylation is generally carried out with T4 polynucleotide kinase and ATP, a reaction that is not particularly reliable or efficient. Several methods for chemical 5'-phosphorylation are also known, including that described in Nadeau et al., Biochemistry 23:6153-6159 (1984), van der Marel et al., Tetrahedron Lett. 22:1463-1466 (1981), Himmelsbach and Pfleiderer, Tetrahedron Lett. 23:4793-4796 (1982), Marugg et al., Nucleic Acids Res. 12:8639-8651 (1984), and Kondo et al., Nucleic Acids Research Symposium Series 16:161-164 (1985). However, most of these methods involve the use of unstable reagents or require extensive modification of standard deprotection and purification procedures. Similar problems have been found with monofunctional and bifunctional 3'-phosphorylating reagents (see Sonveaux, supra, at 297).
The present invention is directed to novel phosphorylating reagents which overcome the limitations of current phosphorylation procedures. As used herein, the term "phosphorylating reagent" encompasses compounds which can phosphorylate a hydroxyl group directly as well as phosphitylating agents which, when coupled with a subsequent oxidation step, can phosphorylate hydroxyl groups indirectly, i.e., in a two-step reaction sequence. The phosphorylating reagents disclosed herein are also useful in the method of the parent application hereto, U.S. Ser. No. 891,789 (now abandoned), which is directed to a method of synthesizing and purifying oligonucleotides substantially free of erroneous sequences. The disclosure of that application is hereby expressly incorporated by reference in its entirety.
The reagents of the present invention, especially the phosphitylating reagents as will be described, are advantageous in that they are easily accommodated by currently available DNA synthesis machines. Also, the phosphorus blocking groups designated herein as Y, Y' or Y', are easily removed during deprotection of the completed oligonucleotide and do not require any additional deprotection steps. Most importantly, the reagents disclosed herein provide for rapid and accurate online monitoring of oligonucleotide synthesis. That is, the present compounds yield a leaving group upon deprotection of the completed oligonucleotide which is readily observable.